Water 2013, 5, 505-524; doi:10.3390/w5020505 OPEN ACCESS
water ISSN 2073-4441 www.mdpi.com/journal/water Article
Microbial Community Structure of a Leachfield Soil: Response to Intermittent Aeration and Tetracycline Addition Janet A. Atoyan 1, Andrew M. Staroscik 2, David R. Nelson 2, Erika L. Patenaude 1, David A. Potts 3 and José A. Amador 1,* 1
2
3
Laboratory of Soil Ecology & Microbiology, 024 Coastal Institute, University of Rhode Island, Kingston, RI 02881, USA; E-Mails:
[email protected] (J.A.A.);
[email protected] (E.L.P.) Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA; E-Mails:
[email protected] (A.M.S.);
[email protected] (D.R.N.) Geomatrix, LLC, Old Saybrook, CT 06475, USA; E-Mail:
[email protected]
* Author to whom correspondence should be addressed; E-Mail:
[email protected]; Tel.: +1-401-874-2902; Fax: +1-401-874-4561. Received: 22 January 2013; in revised form: 3 April 2013 / Accepted: 15 April 2013 / Published: 25 April 2013
Abstract: Soil-based wastewater treatment systems, or leachfields, rely on microbial processes for improving the quality of wastewater before it reaches the groundwater. These processes are affected by physicochemical system properties, such as O2 availability, and disturbances, such as the presence of antimicrobial compounds in wastewater. We examined the microbial community structure of leachfield mesocosms containing native soil and receiving domestic wastewater under intermittently-aerated (AIR) and unaerated (LEACH) conditions before and after dosing with tetracycline (TET). Community structure was assessed using phospholipid fatty acid analysis (PLFA), analysis of dominant phylotypes using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE), and cloning and sequencing of 16S rRNA genes. Prior to dosing, the same PLFA biomarkers were found in soil from AIR and LEACH treatments, although AIR soil had a larger active microbial population and higher concentrations for nine of 32 PLFA markers found. AIR soil also had a larger number of dominant phylotypes, most of them unique to this treatment. Dosing of mesocosms with TET had a more marked effect on AIR than LEACH soil, reducing the size of the microbial population and the number and concentration of PLFA markers. Dominant phylotypes decreased by ~15% in response to TET in both treatments, although the AIR treatment retained a higher number of phylotypes than the LEACH treatment. Fewer than 10% of clones were common to both
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AIR and LEACH soil, and fewer than 25% of the clones from either treatment were homologous with isolates of known genus and species. These included human pathogens, as well as bacteria involved in biogeochemical transformations of C, N, S and metals, and biodegradation of various organic contaminants. Our results show that intermittent aeration has a marked effect on the size and structure of the microbial community that develops in a native leachfield soil. In addition, there is a differential response of the microbial communities of AIR and LEACH soil to tetracycline addition which may be linked to changes in function. Keywords: PLFA; PCR-DGGE; domestic wastewater; intermittent aeration; tetracycline
1. Introduction An understanding of how microbial communities respond to changes in physicochemical conditions and disturbances is necessary for effective development and management of innovative soil-based wastewater treatment systems. Although microorganisms are universally acknowledged as key components in the treatment of septic tank effluent (STE) in soil-based systems, information about the size, structure and function of these microbial communities—and their response to changes in environmental conditions—is scant. This is in contrast with biological processes in centralized wastewater treatment plants, to which state-of-the-art molecular techniques have been applied to elucidate the structure and function of the microbial communities involved in wastewater renovation for some time [1]. Early studies examining microbial populations of soil absorption systems employed culture-based methods [2,3]. Culture-based analyses of the microbial community, although a useful first step, provide limited information, since only a fraction of the community—that amenable to growth under the conditions provided – can be analyzed using this approach [4]. Culture-based analyses of microbial communities can lead to erroneous conclusions regarding the importance of particular organisms in treatment processes and thus ineffective or counterproductive recommendations for their optimization. Amador et al. [5] employed molecular techniques to examine the microbial community structure of soil-based treatment systems using mesocosms filled with synthetic sand. Phospholipid fatty acid (PLFA) and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses indicated that intermittent aeration affected the size and structure of the microbial community. Proteobacteria and actinomycetes/sulfate-reducing bacteria constituted a higher proportion of the community in the aerated treatment, whereas anaerobic Gram-negative bacteria/firmicutes were more prominent in the unaerated treatment. In addition, higher species richness was found in the aerated treatment. The marked effects of intermittent aeration on community structure of soil-based treatment systems are likely linked with improvements in water quality (e.g., BOD, nutrient and pathogen removal) resulting from aeration [6]. More recently Tomaras et al. [7] used 16S rDNA gene sequence analysis to assess microbial community diversity in onsite wastewater treatment systems (OWTS). They reported strong differences in community composition among septic tank effluent, the biomat at the infiltrative surface, and soil that had not received STE. Furthermore, there was no overlap of
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sequences between STE and biomat communities, with considerably less phylogenetic diversity in the latter. In the present study we describe the results of a mesocosm-scale study at an OWTS research facility using mesocosms filled with native soil to simulate conventional and intermittently aerated soil treatment areas. STE amended with tetracycline (TET) was used to regularly dose the lysimeters for a period of 10 days. Tetracycline was chosen as the antibiotic for evaluation because: (i) it has been shown to persist in the environment by adsorbing to soils [8,9]; (ii) it is a broad-spectrum antibiotic used in human medicine that is effective against both Gram-negative and Gram-positive bacteria [10]; and (iii) several of its degradation products also have antibiotic activity [11]. The soil microbial community was characterized using PLFA analysis, PCR-DGGE, and cloning followed by 16S rDNA gene sequence analysis. Differences in community structure were examined between aerated and unaerated soil before the addition of TET, and in response to TET addition for each treatment. 2. Materials and Methods 2.1. Experimental Facility The study was conducted at a research facility in southeastern Connecticut, USA built adjacent to a two-family home fitted with a conventional septic system. Three to six people inhabited the home continuously during the study. A detailed description of the facility can be found in Potts et al. [6]. To the best of our knowledge, none of the residents was taking antibiotics during the course of our study. Septic tank effluent was diverted to a high-density polyethylene (HDPE) storage tank (1325 L) above the laboratory in a climate-controlled room (17–19 °C) (Figure 1). STE from the storage tank was pumped every 6 h (3:00 a.m., 9:00 a.m., 3:00 p.m. and 9:00 p.m.) to dosing tanks in the laboratory. Levels of dissolved organic carbon in STE ranged from 71 to 121 mg C L−1. The dose flowed by gravity from these tanks into mesocosms consisting of stainless steel lysimeters (35.6 cm i.d., 61 cm height) filled with a mixture of B and C horizon soil from a sandy-skeletal, mixed, mesic Typic Udorthent (particle size distribution: 92% sand, 8% silt), representative of soil used in OWTS construction in the southern New England, USA region. The soil was homogenized using a cement mixer prior to use. The remaining space constituted the headspace. The dose was delivered to the soil surface through a horizontal PVC pipe in which holes were drilled. The bottom of the mesocosms was filled with 7.5 cm of No. 4 silica sand overlaid with 30 cm of native soil. The mesocosms began receiving wastewater on 13 August 2003 at a rate of 4 cm day−1. On 22 June 2004, this rate was increased to 12 cm day−1, remaining constant for the duration of the experiment. 2.2. Aeration The headspace of mesocosms was either vented to the septic system leachfield of the house to simulate a conventional leachfield atmosphere (LEACH treatment) or was aerated intermittently with ambient air (AIR treatment) using a process that has been employed successfully to rejuvenate hydraulically-failed septic systems [12]. Each treatment was replicated three times. Air was pumped at regular intervals into the headspace of the AIR mesocosms to maintain O2 levels close to atmospheric (~0.21 mol mol−1) (Figure 1).
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Figure 1. (a) Schematic diagram of laboratory facility and (b) leachfield mesocosms employed in this study. Drawings are not to scale (after Patenaude et al. [13]) Mixing Pump
Effluent Sample Port
Effluent From Holding Tank
Effluent Holding Tank
Dosing Tank
From Holding Tank
PVC Manifold
Solenoid Valve Headspace Gas Sample Port
Dosing Tank
Inspection Port
Solenoid Valve
SOIL
Lysimeter SAND
(a)
Sample Port
(b)
2.3. Antibiotic Dosing Mesocosms were dosed with STE amended with tetracycline (final conc. = 5 mg L−1) every 6 h for 10 days, beginning on 13 June 2005 at 3 p.m. (Day 0). The rationale for antibiotic dosing along with wastewater properties, are described in Patenaude et al. [13] and Atoyan et al. [14]. To amend the wastewater with TET, an aqueous stock solution (500 mg tetracycline HCl L−1; CAS 64-75-5, Sigma Aldrich, Saint Louis, MO, USA) was prepared and kept at ~8 °C in an insulated container packed with ice and equipped with an IceProbe® thermoelectric water chiller (Coolworks®, San Rafael, CA, USA). A peristaltic pump (Thomas Scientific, Swedesboro, NJ, USA) was actuated by a solenoid valve to deliver ~28 mL of TET stock solution to the horizontal PVC pipe within the lysimeters (Figure 1) every 6 h, coincident with wastewater dosing. This mixed the antibiotic stock solution with the wastewater as it flowed into the lysimeters. 2.4. Soil Sampling Soil samples (4-cm deep) were collected on Days 0 and 11. Approximately 4 h prior to the 3 p.m. dosing event the access port was opened, and STE on the soil surface of the LEACH mesocosms was removed by siphoning and stored. No STE had accumulated on the soil surface of AIR mesocosms, thus there was no need for removal. Five soil cores (2.75-cm dia., 4-cm height) were taken aseptically from each mesocosm using cut-off, 60-mL plastic syringes. STE was returned to the mesocosms after soil sampling. Soil cores were placed in sterile Whirl-Pak® bags and kept on ice during transport to the laboratory. Immediately upon returning to the laboratory, 50 g of homogenized soil from each mesocosm was shipped on ice by overnight courier to Microbial Insights, Inc. (Rockford, TN, USA) for PLFA analysis. The remaining soil was stored at −80 °C for subsequent analysis.
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2.5. Phospholipid Fatty Acid Analysis PLFAs were extracted using a modification [15] of the method of Bligh and Dyer [16], with one soil sample analyzed per mesocosm. Fatty acid methyl esters were separated by gas chromatography and identified by retention time and mass spectrometry as described by Tunlid et al. [17]. The detection limit was 7 pmoles of PLFA. For the purpose of community structure analysis, PLFAs were divided into markers for six different microbial groups [18–21]: (i) firmicutes/anaerobic Gram-negative bacteria, (ii) proteobacteria, (iii) anaerobic metal reducers, (iv) sulfate-reducing bacteria (SRB)/actinomycetes, (v) general bacteria, and (vi) eukaryotes. 2.6. DNA Extraction from Soil DNA was extracted from ~1 g homogenized soil from each mesocosm using the bead-beating UltraClean Soil DNA Isolation kit (MoBio, Carlsbad, CA, USA) per manufacturer’s instructions. DNA was further purified by spin-column chromatography following the protocol for BD Chroma Spin + TE-100 columns (Clontech, Mountain View, CA, USA), and concentrated by ethanol precipitation and resuspension in 20 μL EB buffer. 2.7. PCR-DGGE Extracted DNA was amplified by polymerase chain reaction (PCR) with the primers 518R (5'-ATT ACC GCG GCT GCT GG-3') and 357F-GC (5'-CCT ACG GGA GGC AGC AGC GCC CGC CGC GCG CGG CGG GCG GGG CGG GGG CAC GGG GGG-3') specific for the 16S rDNA gene of bacteria, modified from Marchesi et al. [22] by the addition of a GC clamp [23]. Four PCR reactions were performed for each replicate mesocosm. PCR was performed using the Taq PCR Master Mix kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol with 10 ng of template DNA per 50 µL reaction. PCR was performed in a GeneAmp thermocycler (Applied Biosystems, Foster City, CA, USA) under the following conditions: initial denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min 30 s, and a final extension at 72 °C for 7 min. PCR products were purified and concentrated using the Qiaquick PCR Purification kit (Qiagen). The products from all four PCR reactions from a mesocosm were applied to one column and quantified using an Ultrospec 4000 spectrophotometer (Pharmacia Biotech, Piscataway, NJ, USA). Approximately 200 ng of PCR product per lane was loaded onto a polyacrylamide gel for generation of community profiles. Electrophoresis was run as described by Muyzer et al. [24] using a CBS Scientific DGGE system (Del Mar, CA, USA) on a 0.75-mm thick, 8% (w/v) polyacrylamide gel with a gradient from 60% to 40% denaturant, where 100% denaturant had a concentration of 7 M urea and 40% (v/v) formamide. The gel was run in 0.5 × TAE buffer for 16 h at 200 V and 60 °C and stained for 30 min in SYBR Green dye. The gel was visualized using a Typhoon 9410 variable mode imager. Bands were identified using ImageJ software [25] with rolling ball subtraction (r = 10). 2.8. Clone Libraries Extracted DNA was amplified by PCR with primers B27f (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1387R (5'-GGG CGG WGT GTA CAA GGC-3'), specific for the 16S rDNA of bacteria [22]. Four
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PCR reactions were performed for each replicate mesocosm. PCR, amplicon purification, and quantification were performed as for PCR-DGGE analysis. Four clone libraries were constructed: one per treatment—AIR and LEACH—for Day 0 and Day 11. Cloning reactions were performed following the standard protocol for the TOPO TA Cloning Kit for Sequencing (Invitrogen, Chicago, IL, USA) using mixed PCR product from each of the three replicates per treatment weighted by the concentration of DNA in each replicate. Approximately 100 colonies were then chosen randomly for sequencing on a Beckman Coulter CEQ 8000 using the primer B27f. Clone library sequences were aligned and chimeric sequences were removed using the NAST alignment tool and Bellerophon [26]. Clones were analyzed for phylogenetic similarity using the Greengenes DNA maximum likelihood (DNAML) classification tool. 2.9. Data Analysis The Dice similarity coefficient, Cs, was calculated as described by Amador et al. [5]. Indices of richness (S) were calculated based on Staddon et al. [27]. Paired t-tests were used to compare the responses of this variable to TET addition (Day 0 vs. Day 11) within a particular treatment. The p value for all analyses was
